Rat brain cytosol contains several distinct proteins that inhibit phospholipase D (PLD) activity. Two of these proteins were previously purified and identified as synaptojanin and AP180, both of which are specific to nerve terminals and associated with the clathrin coat. Two additional PLD-inhibitory proteins were purified and identified as amphiphysins I and II, which are known to form a heterodimer that associates with the clathrin coat. To determine whether AmphI forms a complex with PLD enzymes in intact cells, we cotransfected COS-7 cells with vectors encoding AmphI and either HA-tagged PLD1 or HA-tagged PLD2. Immunoprecipitation experiments with anti-HA antibodies followed by blotting with antibodies to AmphI demonstrated that AmphI tightly associates with PLD1 and PLD2 in intact cells. The effects of amphiphysins on PLD activity in intact cells were also investigated by transient expression of AmphI or AmphII in COS-7 cells. The PMA- stimulated PLD activity was reduced by ~30% in cells expressing AmphI or AmphII , suggesting that endogeneous PLD activity was inhibited by the exogenous amphiphysins. This relatively low percentage inhibition, given the marked inhibitory efficacy and potency of AmphI and AmphII apparent in vitro, is likely attributable to the fact that only a fraction (usually 20 to 40%) of the cells is actually transfected To overcome the low transient transfection efficiency, we prepared a retrovirus vector harboring AmphI cDNA. Infection of baby hamster kidney (BHK) cells with this vector revealed that the PMA-stimulated activity of endogenous PLD was inhibited by the expression of AmphI in a dose-dependent manner, with virtually complete inhibition apparent at the highest tested dose of the vector. Expression of recombinant amphiphysins in COS-7 cells resulted in inhibition of both endogenous PLD activity as well as that of exogenously expressed PLD1 or PLD2. The NH2-terminal 373 amino acids, but not the COOH-terminal 404 residues, of amphiphysin I were critical for both inhibition of and binding to PLD. Phosphatidic acid, a product of PLD-catalyzed hydrolysis of phosphatidylcholine, is thought to be required for the assembly of clathrin-coated vesicles during endocytosis. Thus, the inhibition of PLD by these various clathrin coat-associated proteins (amphiphysins, synaptojanin, and AP180) might play an important role in synaptic vesicle trafficking. - amphiphysin/clathrin/endocytosis/phospholipase D/synaptic terminal